Project Summary R-bodies are self-assembling protein polymers from bacterial ensymbionts of 'killer' Paramecium strains that confer to the host the ability to kill competing strains. The tightly coiled sub-micron sized R-body undergoes a massive pH induced conformational change into elongated rods tens of microns long, generating forces sufficient to rupture biological membranes. Purified recombinant R-bodies can undergo many successive rounds of piston-like extension and contraction using only the chemical energy from pH change. These robust nanomachines have enormous potential for biotechnological and therapeutic applications. Their ability to behave as sensitive switches and to do work on biological structures can be adapted for many functions - they have, for example, been proposed as systems for phagosomal delivery of bioactive molecules in a way that would mimic their biological function. The ability to tune R-body behavior was recently demonstrated by a panel of point mutants that alter the triggering pH for extension. However, the lack of detailed molecular understanding of their assembly and the mechanism of the pH-sensitive piston-like extension are major roadblocks to developing R-bodies for novel applications. This proposal aims to determine the molecular architecture of R-bodies using a hybrid structural approach, and to measure their biophysical properties using optical trapping force measurements. Visualizing the three-dimensional structure of R-bodies in coiled and extended states using cryo-EM will allow us to define the molecular mechanism of their piston-like behavior, laying the groundwork for tuning the structures to new functions. This request for administrative equipment supplement is to upgrade our Titan Krios cryo-electron microscope with ?fringe-free? illumination, a proven strategy for increasing the throughput of cryo-EM data collection, which will allow us to collect larger datasets faster, greatly expanding the capacity of our existing instrumentation.